Advances in MRI using the contrast agent Gadolinium (Gd) have led to a greatly enhanced precision in diagnostics. It has not yet been possible to depict the cell itself due to the extracellular distribution characteristics of the commonly used Gadolinium contrast agents. The intensively discussed and investigated Molecular Imaging methods could open the door to imaging at the cellular level.
In order to depict the cell, a contrast agent is required which can pass into the intracellular space. There have been numerous proposals as to how this could be achieved: It was attempted to achieve an optimal uptake of iron in the cell using a conventional non-viral transfection method to promote the expression of the transferrin receptor.
The gene for the transferrin receptor was transfered with the help of an adenovirus. However, it became apparent that the cells which overexpressed the transferrin receptor protected themselves from excess iron concentrations via an mRNA-mediated negative feedback inhibition of the transferrin-receptor causing decreased iron influx. This problem was overcome using ETfR (Engineered Transferrin Receptor) and MIONs (Micro Iron Oxide Nanoparticles). This process is based on liquid-phase-endocytosis of dextrane-coated MIONs via the transferrin receptor.
A further potentially attractive method for molecular imaging is the use of Gadolinium complexes (Magnevist® Schering). It has been shown that the commonly used contrast agent Magnevist® is very well suited to the display of the intercellular space, but is not suitable for intracellular imaging.
Micro-injection methods were used in Xenopus Laevis embryos (2-cell stadium) in order to accumulate gadolinium successfully in the intracellular space. One group attempted to accumulate a Gd-complex in the cell utilising high extracellular concentrations of a Gadolinium-complex (1-25 mg/ml) in which maximal intracellular concentrations were attained after 100 hours. With the help of a viral transporter (HIV-1 tat-peptide) high intracellular concentrations of gadolinium and iron oxide nanoparticles were achieved. Another group has even identified the HIV-1 tat peptide in the cell nucleus. There are, however, still open questions as to the transactivating effects of the viral transporter HIV-1 tat peptide in the nucleus such as the induction of apoptosis in hippocampal neurons. To summarize, there are serious disadvantages of the previous approaches, e.g., the incubation time for, e.g., the Gadolinium-complex is far too long and the concentration of this complex that has to be used is extremely high resulting in serious side effects. Moreover, despite the advances in cellular transport, there remains the question of cell specificity, i.e. all the above mentioned methods have one problem in common: they cannot differentiate between tumor and non-tumor cells.
Therefore, it is the object of the present invention to provide a diagnostic means which overcomes the disadvantages of the diagnostic tools of the prior art, i.e. which allows the fast and precise non-invasive determination, preferably the molecular imaging, of gene expression pattern in cells of a patient.